Tetherin (BST2, CD317 or HM1.24) was recently identified as an interferon-inducible host-cell factor that interferes with the detachment of virus particles from infected cells. HIV-1 overcomes the antiviral effects of tetherin by expressing Vpu, which downregulates and degrades tetherin. Simian immunodeficiency virus (SIV) does not have a vpu gene. We therefore set out to determine how SIV overcomes this restriction in the rhesus macaque, an important animal model for lentiviral pathogenesis and for AIDS vaccine development. We found that the SIV Nef protein could counteract rhesus tetherin, but not human tetherin. Conversely, the HIV-1 Vpu protein counteracted human tetherin, but not rhesus tetherin. These observations identified a role for Nef in antagonizing tetherin in the rhesus macaque, and revealed species-specificity in the activities of both SIV Nef and HIV-1 Vpu in overcoming tetherin in their respective hosts. Here we propose to define alternative mechanisms for overcoming restriction by tetherin in the rhesus macaque and to adapt HIV-1 for replication in cells expressing rhesus tetherin. Previous studies have demonstrated that infection of rhesus macaques with pathogenic derivatives of nef- deleted SIV vaccine strains, and with SIV recombinants expressing nef alleles of HIV-1 (SHIVnef), can result in persistent viral loads and progression to AIDS. We now have preliminary data to suggest that these viruses have acquired genetic changes in their env and nef genes that confer resistance to rhesus tetherin. To investigate this hypothesis, we will define sequence changes selected in SIV nef- and SHIVnef-infected macaques that afford resistance to tetherin (Specific Aim 1). Species-specific differences in tetherin also represent a potential additional barrier to HIV-1 replication in macaques. The introduction of sequence changes into simian-tropic strains of HIV-1 that enhance the resistance of these viruses to rhesus tetherin therefore represents a promising approach for further adapting HIV-1 for replication in rhesus macaques. To adapt HIV-1 for replication in cells expressing rhesus tetherin, we will introduce nef alleles derived from SHIVnef-infected animals, or from HIV-1 and SIVcpz isolates found to have activity against rhesus tetherin, into a simian-tropic strain of HIV-1 (Specific Aim 2). The significance of this proposal is two-fold. First, these studies are expected to reveal alternative mechanisms for overcoming restriction by tetherin fundamental to our understanding of lentiviral pathogenesis. Second, these studies are expected to facilitate the development of a macaque model for HIV-1 infection. PUBLIC HEALTH RELEVANCE: The studies outlined in this proposal are expected to reveal fundamental mechanisms by which HIV-1 and SIV overcome inhibition by tetherin, an innate host-cell defense against viral pathogens, and to facilitate the development of a non-human primate model for HIV-1 infection.